Protein-Polymer Conjugates (PPCs) have been described as a marriage between two diverse yet symbiotic materials. PPCs combine the biological specificity of proteins with the diverse functions and tunable properties of synthetic polymers. Proteins and other biologically active molecules are used in a number of diagnostic, monitoring or treatment applications by virtue of their biological activity and specificity. However the in vivo delivery of such agents can encounter several limitations including low solubility, poor stability, short half lives and a potential to create an immunogenic response which may require frequent administration of the agent or generate a risk of adverse reactions. The conjugation of proteins with well defined polymers allows the resulting PPC to overcome some of the inherent limitations of specific proteins targeting specific biological applications.
When one takes into consideration the rate at which reversible-deactivation radical polymerization (RDRP), formerly named controlled radical polymerization (CRP) procedures that include Nitroxide Mediated Polymerization (NMP), Atom Transfer Radical Polymerization (ATRP), and Reversible Addition Fragmentation Transfer (RAFT) have evolved over the last two decades, to provide access to (co)polymers with predefined molecular weights, compositions, architectures and narrow/controlled molecular weight distributions, it is no surprise that a growing number of researchers have decided to combine the biological specificity of proteins and other biologically active (macro)molecules, including peptides, nucleic acids, and carbohydrates, with the diverse functions and tunable properties of synthetic polymers prepared by RDRP by conjugation to form well-defined polymer protein hybrids (PPH); a procedure frequently termed bioconjugation. [Curr. Opin. Chem. Biol., 2010. 14(6): 818-827] Functionalization of bioresponsive molecules with well defined polymers can provide improved stability, tailored solubility, predeterminable trafficking pathways, and increased therapeutic potential of already useful biomacromolecules including peptides, proteins, nucleic acids, and polysaccharides in a variety of applications.
Indeed, since RDRP processes can provide compositionally homogeneous well-defined polymers, with predicted molecular weight, narrow molecular weight distribution, and high degrees of α- and ω-end-functionalization, they have been the subject of much study as reported in several review articles and ACS symposia. [Matyjaszewski, K., Ed. Controlled Radical Polymerization; ACS: Washington, D.C., 1998; ACS Symposium Series 685. Matyjaszewski, K., Ed.; Controlled/Living Radical Polymerization. Progress in ATRP, NMP, and RAFT; ACS: Washington, D.C., 2000; ACS Symposium Series 768; Matyjaszewski, K., Davis, T. P., Eds. Handbook of Radical Polymerization; Wiley: Hoboken, 2002; Qiu, J.; Charleux, B.; Matyjaszewski, K. Prog. Polym. Sci. 2001, 26, 2083; Davis, K. A.; Matyjaszewski, K. Adv. Polym. Sci. 2002, 159, 1.]
Matyjaszewski and coworkers disclosed the fundamental four component ATRP process comprising the addition, or in situ formation, of an initiator, in this case a molecule with a transferable atom or group that is completely incorporated into the final product, a transition metal and a ligand that form, a partially soluble transition metal complex that participates in a reversible redox reaction with the added initiator or a dormant polymer to form the active species to copolymerize radically polymerizable monomers, and a number of improvements to the basic ATRP process, in a number of commonly assigned patents and patent applications: U.S. Pat. Nos. 5,763,546; 5,807,937; 5,789,487; 5,945,491; 6,111,022; 6,121,371; 6,124,411; 6,162,882; 6,624,262; 6,407,187; 6,512,060; 6,538,091; 6,541,580; 6,624,262; 6,627,314; 6,759,491; 6,790,919; 6,887,962; 7,019,082; 7,049,373; 7,064,166; 7,125,938; 7,157,530; 7,332,550; 7,407,995; 7,572,874; 7,678,869; 7,795,355; 7,825,199; 7,893,173; 7,893,174; U.S. Ser. Nos. 12/877,589; 12/949,466; and International patent applications PCT/US04/09905; PCT/US06/33152; PCT/US06/048656; PCT/US08/64710; PCT/US09/36377; PCT/US2010/029073; PCT/US2011/051043 and PCT/US11/65578 all of which are herein incorporated by reference to provide background and definitions for the present disclosure.
The generally accepted mechanism of an ATRP reaction is shown in Scheme 1.

ATRP is the most efficient RDRP method for the preparation of pure segmented copolymers, since, unlike RAFT, it does not require addition of a radical initiator to continuously form new polymer chains that do not contain the desired α-biofunctional group in a grafting from reaction and unlike NMP does not require high temperatures to generate the active species by homolytic cleavage of the dormant chain end. ATRP allows the synthesis of new telechelic multi-segmented copolymers with a predetermined degree of polymerization, low molecular weight distribution (Mw/Mn, also called polydispersity index or PDI), incorporating a wide range of functional monomers and displaying controllable macromolecular structures under mild reaction conditions. ATRP generally requires addition or formation of an alkyl halide or (pseudo)halide as an initiator (R—X) or dormant polymer chain end (Pn—X), and a partially soluble transition metal complex (Cu, Fe or Ru, for example) capable of undergoing a redox reaction as a catalyst. This procedure would be expected to be ideal for the preparation of PPCs but surprisingly, considering the prior efforts in this field there have been problems preparing well defined bioconjugate materials.
As noted above PPCs have a relatively long history of being used therapeutically because they exhibit the properties of both the biomolecules and the polymer, with the polymer actively providing a means to tune the bioactivity of the biomolecule or passively stabilizing the biomolecules in vivo to allow an increase in blood circulation time or improved tumor targeting by enhanced permeation and retention effects. The primary applications of PPCs have been developed by the pharmaceutical industry, where they are used as highly potent therapeutics. PPCs have also been applied to prepare biological sensing devices. The first generation of PPCs were described in 1977 by Abuchowski et al. [J. Biol Chem. 1977, 252, 3578 & 3582] The authors reported that proteins modified with poly(ethylene glycol) (PEG) have a greatly increased in vivo circulation time and reduced immunogenicity, and this started an extended study of PEG based bioconjugation because PEG and its derivatives are nontoxic, non-immunogenic, and possess biocompatible properties.
Recently there has been considerable interest dedicated to alternative methods of “PEGylation” by the polymerization of PEG or oligo(ethylene glycol) (OEG) vinyl-macromonomers. Many of these methods allow the preparation of polymers that are both biocompatible and stimuli-responsive. Increasingly, polymer bioconjugates with therapeutic potential are being made with synthetic polymers other than PEG based copolymers. This work has given rise to an expanding research field where stimuli responsive polymers are conjugated to bioresponsive molecules [Nat. Rev. Drug Discovery 2003, 2, 347; Bioorg. Med. Chem. 2007, 15, 4382; J. Am. Chem. Soc. 2010, 132, 13575]. Similar to PEG, these polymers may also enhance the stability and solubility of the biological component to which they are attached, while simultaneously providing responsive behavior and numerous sites for subsequent functionalization; e.g., to allow the attachment of cofactors, targeting ligands, imaging reagents, etc. The synergistic effect of polymer bioconjugation can be further enhanced by the introduction of functional groups into the polymer's backbone. New drug delivery systems can be envisioned where hydrophobic drugs are encapsulated into a PPC above the polymers lower critical solution temperature (LCST) and released in vivo in a predetermined area at a predeterminable rate.
Two methods are generally used for the preparation of PPCs: the “grafting to” approach, which involves conjugation of a preformed polymer to a protein, and the “grafting from” approach, which involves growing a polymer from a known initiator site within the protein.
“Grafting to” has been used to conjugate a variety of polymers to proteins including poly(ethylene glycol), thermo-responsive polymers, drug-loaded polymers, and dye-loaded polymers among others. Indeed due to the ongoing development of bio-orthogonal “click chemistry” methods [Angew. Chem. Int. Ed., 2001. 40(11): 2004]. Bioconjugation has furthermore become increasingly selective, which has led to hybrid structures with improved functional characteristics. While RDRP techniques have allowed synthesis of precisely defined PPCs with properties unachievable through simple PEGylation and the procedure allows for the straightforward formation of, and subsequent conjugation of, polymers to proteins, under high yield conditions “grafting to” has several limitations. Typically PPC's prepared by “grafting to” suffer from substantial batch-to-batch variability and generate a broad distribution of products. This variability is due to the presence of a multiplicity of reactive groups on the surface of a targeted protein in addition to steric constraints generated when grafting two large macromolecules together. Furthermore, purification of the desired PPC from a multitudinous mixture of modified proteins and free polymer is challenging.
The alternative approach to synthesizing PPC's is the “grafting from” method. In this method an initiating group is immobilized onto a protein, typically through acetylation of lysine or cysteine residues present within the protein, and a polymer is grown from the incorporated initiator/control agent in situ. The major advantages of protein polymer conjugates formed by “grafting from” procedures are high yields and avoidance of traditional purification issues associated with the “grafting to” method. The field of using controlled radical procedures for a “grafting from” has been slowly gaining popularity as a standard tool to creating functional PPC's.
In the first example of this method, a biotinylated ATRP initiator was bound to streptavidin and PNIPAM or POEOMA chains were grown from the streptavidin tetra initiator [J. Am. Chem. Soc. 2005, 127, 6508; U.S. Pat. No. 7,786,213 B2]. In a later work, POEOMA was grown from the C-terminus of green fluorescent protein (GFP) and shown to accumulate in vivo in a tumor mouse model [Proc. Natl. Acad. Sci. 2010. 107(38): 16432-7; and PCT Publication WO 2010/096422]. More recently, an engineered non-natural amino acid bearing an ATRP initiating site was genetically incorporated into the 134 amino acid residue of GFP [J. Am. Chem. Soc. 2010, 132, 13575] and POEOMA was grown from this genetically encoded initiator. The retention of the fluorescence demonstrated preservation of the GFP's native tertiary structure during the RDRP. This initial work on preparation of a protein-polymer conjugate (PPC) based on a RDRP, specifically a “grafting from” ATRP, provided a composite structure with a stable link between the protein and the copolymer was disclosed in U.S. Provisional Application 61/381,757 filed on Sep. 10, 2010, converted to PCT/US2011/051043, which is hereby incorporated by reference.
However, achieving a high degree of control over the “grafting from” processes has proven to be challenging. Typically the gel permeation chromatography (GPC) curves of the formed PPC do not display a normal distribution of molecular weights and provide high values for molecular weight distribution (Mw/Mn). The curves observed in size exclusion chromatography display a substantial tailing to low the molecular weight region, which would indicate low and/or poor initiation efficiencies. As noted in the PCT/US2011/051043 application, specifically in the discussion relating to FIG. 7C of the '043 application, reproduced herein as FIG. 1, there is a tailing towards low Mn region of the GPC elutogram of the product formed during the grafting from reaction, compare the dark line for parent GFP with the grey line from the product. This extended tailing indicates the formation of non-uniform protein-polymer conjugates that could have negative implications for controlled therapeutic protein delivery and uniform enzymatic processes. This is not a unique observation since a similar problem is seen in other published GPC traces from PPCs formed by “grafting from” reactions; FIG. 10 in U.S. Pat. No. 7,786,213 and FIGS. 10, 13 and 26 in PCT application WO 2010/096422 and, indeed in any prior art work conducted under previously envisioned bio-compatible conditions.
Therefore one of the remaining challenges present when seeking to incorporate a protein, or other biologically responsive molecule, into a well defined protein-polymer conjugate in a “grafting from” reaction is to identify, define, and exemplify conditions for the “grafting from” reaction that provides uniform well defined tethered chain(s). A target for the degree of dispersity of the tethered copolymer chain that is accepted as indicative of good control in a RDRP grafting from reaction is a Mw/Mn of less than 1.30, preferably less than 1.25 and more preferably less than 1.20. In addition the reaction should not affect the properties of the biologically responsive molecule; thereby providing a bioconjugate in which uniform (co)polymer segment(s) are attached to the protein, or other biologically responsive molecule, at known sites within the bioresponsive molecule that do not modify the physiological action of the protein or other biologically responsive molecule in an undesired fashion, or reduce the effectiveness of the desired bioresponsive action to any significant degree.
To summarize the current state of the art focusing on “grafting from” proteins has resulted in a situation where reaction conditions utilized for the “grafting from” reaction are copied from conditions employed for a biologically inactive initiator, frequently a small molecule. The conditions are taken from the literature then applied to a new bio-sensitive macroinitiator. This means that a wide range of polymerization conditions have been used to prepare PPCs including a variety of different monomer and protein-initiator concentrations, catalyst to initiator ratios, catalyst systems, i.e. ligands and copper halides, copper(I) to copper(II) ratios, and solvent systems; essentially procedures where reagents are mixed with a protein initiator and the reaction is stopped after a seemingly random time frame. Despite claims for a successful grafting from protein polymers conjugates produced by these procedures have long tail towards low molecular weights and there has been little description of the rate of the polymerization with respect to conversion or time [J. Am. Chem. Soc. 2005, 127(18), 6508-6509; Biomacromolecules 2005, 6(6), 3380-3387; Angew. Chem. Int. Ed. 2008, 47(33), 6263-6266; Proc. Natl. Acad. Sci. USA 2009, 106(36), 15231-6; Proc Natl Acad Sci USA 2010, 107(38), 16432-7; Adv. Drug Deliv. Rev., 2010. 62(2): 272-82].
Other RDRP techniques have also been examined, on the basis that ATRP requires a high concentration of an undesireable metal catalyst, include RAFT [Macromol Rapid Commun. 2011, 32, 354] and NMP [Polym. Chem., 2011. 2(7): 1523-1530]. These procedures also possess inherent limitations when targeting well defined PPCs. In the case of RAFT a secondary source of radicals is required to drive the reaction thereby forming non-conjugated polymer contaminants. NMP requires a high temperature to form the active propagating radical which can denature the bio-active agent. In common with prior art ATRP procedures the products of both procedures displayed a broad dispersity; Mw/Mn=≤1.40 and Mw/Mn=≤1.33 respectively.
In order for a “grafting from” procedure to become a widely utilized method in the preparation of PPCs the aforementioned challenges must be addressed and conditions that lead to direct preparation of a well defined PPC must be established. Preparation of these materials under biologically compatible conditions would allow synthesis of biomaterials in their native environments and minimize post fabrication purification steps while preserving biological activity.